1. Field of the Invention
The invention relates generally to a purification protocol for obtaining a low molecular weight, thermostable .beta.-D-glucosidase from Acidothermus cellulolyticus The present application incorporates by reference the entirety of U.S. patent application Ser. No. 08/125,115. In particular, the invention pertains more specifically to a process for obtaining a low molecular weight, thermostable .beta.-D-glucosidase from Acidothermus cellulolyticus culture broth or bacterium, submitted to the American Type Culture Collection under collection number 43068. The address where the Acidothermus cellulolyticus was deposited is 12301 Parklawn Drive, Rockville, Md. 20852.
Cellulose consists of long insoluble chains of covalently bonded glucose molecules, and in this condition, these long insoluble chains are too large to be transported through human and animal cell walls. However, through the agency of microorganisms, such as fungi and bacteria, enzymes known as cellulases are secreted, and these enzymes hydrolyze or depolymerize the cellulose into its monomeric component of glucose, which is a sugar that can be readily transported through the cell wall and metabolized.
The fermentable fractions of cellulosic biomass include cellulose (.beta.-1,4-1inked glucose) and hemicellulose, a substantial heterogeneous fraction that is composed of xylose and minor five- and six-carbon sugars. Although it is an abundant biopolymer, cellulose is unique in that it is highly crystalline, insoluble in water, and highly resistant to depolymerization. The definitive enzymatic degradation of cellulose to glucose (the most desirable fermentation feedstock), is generally accomplished by the synergistic action of three distinct classes of enzymes: first, the "endo-1,4-.beta.-glucanases" or 1,4-.beta.-glucan 4-glucanohydrolases (EC 3.2.1.4), which act at random on soluble and insoluble 1,4-.beta.-glucan substrates and are commonly measured by the detection of reducing groups released from carboxymethylcellulose (CMC); second, the "exo-1,4-.beta.-glucosidases," including both the 1,4-.beta.-glucan glucohydrolases (EC 3.2.1.74), which liberate D-glucose from 1,4-.beta.-glucans and hydrolyze D-cellobiose slowly, and 1,4-.beta.-D-glucan cellobiohydrolase (EC 3.2.1.91), which liberates D-cellobiose from 1,4-.beta.-glucans; and third, the ".beta.-D-glucosidases" or .beta.-D-glucoside glucohydrolases (EC 3.2.1.21), which act to release D-glucose units from soluble cellodextrins and an array of glycosides. Synergistic actions of these three enzymes are necessary to completely depolymerize cellulose into glucose.
The synergistic reaction occurs as a result of a sequential, cooperative action among the three enzyme components in a complex in which the product of one enzyme reaction becomes the substrate for the next enzyme.
The development of economic processes for the conversion of low-value biomass to ethanol via fermentation requires the optimization of several key steps, especially that of cellulase production. This condition results from the extraordinarily high ratios of cellulase required to fully depolymerize cellulose. The problem is compounded by the relatively slow growth rates of cellulase producing fungi and the long times required for cellulase induction. However, the product of most cellulase systems is cellobiose, a dimer of glucose. Because cellobiose is almost universally inhibitory to cellulase enzymes, and because .beta.-D-glucosidases convert cellobiose to glucose, the inclusion of .beta.-D-glucosidases in processes where cellulases are used significantly enhances the effectiveness of the overall process of cellulose hydrolysis.
However, industrial processes utilizing cellulase enzymes such as .beta.-D-glucosidase enzyme can be greatly improved and expanded by providing a .beta.-D-glucosidase enzyme with increased thermal resistance for use in high temperature processes. Such resistance would also be useful to ensure increased stabilization resistance under other conditions known to denature enzymes, such as shear stress (from pumping), protease attack, and reduced contamination because they remain active at high process temperatures, which usually destroy other potentially contaminating enzymes.